The need for novel, more efficient and safer drugs capable of addressing the growing issue of multidrug-resistant tuberculosis (MDR-TB) and other antimicrobial resistances call for strategies with the ultimate objective of identifying leads showing favorable physicochemical properties, potency and toxicity. A successful strategy is to build on well-established and first line drugs with poorly understood mechanisms of action or synergy. In this proposal we focus on pyrazinamide (PZA), a drug that is synergistic with nearly all other anti-TB drugs and is included in current anti-TB regimens as a means of reducing the treatment time from nine to six months. However, the mode of action of this drug remains controversial, and no data explains the mechanism behind the powerful synergism between this drug and others. Recent studies have proposed a role in inhibition of trans-translation and/or disruption of bacterial ATP synthesis. Our work with an anti-tubercular in phase 2 clinical trials, SQ-109, and analogs led us to conclude that disruption of proton motive force (PMF), in addition to enzyme inhibition, is important to the efficacy of these compounds. Thus, we hypothesize that disruption of PMF plays a significant role in the synergistic properties of PZA. In this project we propose to dissect the reported modes of action of PZA and a series of related compounds with the goal of determining the relative importance of PMF disruption and trans-translation inhibition in the synergistic response. The results will potentially translate into the development of new and novel lead compounds that are designed to be synergistic with existing and new drugs.